PROJECT SUMMARY/DESCRIPTION Polymerase theta (Pol q, gene name Polq) is essential in many hereditary breast cancers, yet loss of Pol q is well tolerated in most normal cells. As a consequence, there has been much excitement in the development of targeted inhibitors of Pol q for cancer therapy. However, we know little about the biological role and mechanism of action for this large, multi-domain factor. It has thus not been possible to reconcile the disparate, apparently context-dependent impacts of Pol q loss on mutation, chromosome aberration, and cell survival, and the safe, effective targeting of Pol q for therapy has been largely frustrated. In overall Aim 1, the mechanism by which full-length Pol q and Pol q domains contribute to repair will be characterized by parallel analysis using the biochemical, structural, genetic and biophysical imaging approaches available to our program. Full pathway reconstitution and visualization is a goal. In Overall Aim 2 we will investigate the cellular contexts that normally engage Pol q. In Overall Aim 3 we will integrate insights gained from these other Aims to develop rationales for safer, effective targeting of Pol q in cancer therapy. The research work will be highly coordinated within the Program Project in a framework with three Core facilities. Substrates, proteins, and experiments will be designed with all Projects and constantly monitored with feedback via Administrative Core A. Protein purification will be supported by Core B, and cell line construction by Core C. The scientific project leaders have complementary expertise: Drs. Dale Ramsden (molecular biology; Project 1), Gaorav Gupta (cancer cell biology; Project 1), Richard Wood (biochemistry, Project 2) Sylvie Doubli (structural biology and mechanism; Project 3), and Eli Rothenberg (biophysics; Project 4). This ensemble of complementary expertise fosters cross-fertilization of ideas beneficial to the whole team, and makes work possible that can only be accomplished by a Program Project grant. This team also already has a long track record of productive collaboration, and within this program project will work effectively and synergistically to accomplish the Program?s goals. The results obtained by this Program Project will provide a fundamental advance in the understanding of the molecular mechanisms underpinning TMEJ, and will pave the way for the design of novel cancer therapy via Pol ? inhibition.